ease of use and therefore optimal for low, mid to high throughput applications

Nextera and Nextera XT sample preparation with mosquito HTS

Nextera and Nextera XT (Illumina, San Diego, USA) sample prep kits are commonly used to prepare DNA libraries. Both Nextera and Nextera XT, use an enzymatic reaction which fragments the DNA and ligates the adapters simultaneously. In order to obtain fragments of the correct size for the sequencing run, it is essential to have a precise and accurate ratio of tagmentation enzyme to DNA. Using mosquito HTS or mosquito HV, the tagmentation step can be both automated and miniaturised to sub-microliter levels, thus reducing the bottleneck in high throughput applications such as single cell analysis.

Researchers from Professor Stephen Quake’s laboratory at Stanford University, see poster, undertook sequencing of 384 cDNAs extracted from a mixture of 5 different bacteria species (6-20 kb); separated into single cells. Only 60 pg of input gDNA was used per each 4 µL library.

Percent of the reads of 3 different large MW (over 10 kb, Coriell Institute) gDNA libraries prepared at 1 μL in 8 replicates, mapping to the given characteristic: human and unique. Only 1 ng of gDNA was used per library. Very low standard deviations show the great reproducibility of the miniaturised-volume Nextera libraries.

Scientists in Dr Debbie Nickerson’s laboratory at the University of Washington-Genome Sciences Center prepared 3 different large molecular weight (over 10 kb) gDNA libraries prepared at 1 μL in 8 replicates, using Nextera library prep kit, see table above and poster. Only 1 ng of gDNA was used per library and the sample prep cost was reduced by 50 fold as compared to the original 50 µL reactions.

Table above – Fraction of the reads of 3 libraries mapped to the given characteristic: human and unique. Low standard deviations and high mapping ability show the reproducibility of the miniaturised Nextera libraries at 1 μL.